Many questions in biology and neuroscience would benefit greatly from a technology that enabled molecular information (e.g., the identities of specific nucleic acids and proteins) to be imaged throughout preserved 3-D specimens (e.g., brain circuits), with nanoscale precision. Accordingly, we developed a fundamentally new approach, published in Science in 2015: in contrast to earlier methods of magnification in light microscopy, which rely on lenses to optically magnify images of cells and tissues, we physically magnify preserved specimens. By synthesizing a swellable polyelectrolyte gel directly within a specimen, mechanically homogenizing the specimen, then dialyzing in water, we could expand tissues by ~4.5x in linear dimension. This method could separate molecules located within a diffraction-limited volume to distances great enough to be resolved with conventional microscopes, resulting in an effective resolution of ~70 nm. We call this novel method expansion microscopy (ExM). Since then, we have made the technology easier to use, creating a version of ExM which we call proExM (protein retention ExM) that uses commercially available chemicals to directly anchor genetically encoded fluorophores or antibody-borne fluorophores to the swellable gel, and validating its ability to preserve nanoscale features in a variety of tissues (accepted at Nature Biotechnology) and extended ExM to the anchoring and expansion of RNA molecules away from one another for nanoscale RNA visualization, which we call ExFISH (accepted at Nature Methods). There is great pent-up demand for a method of nanoscale imaging for extended 3-D specimens, especially one that requires no specialized equipment; we host visitors weekly in our group at MIT to come and learn and practice ExM, and with the Janelia Research Campus we will run a workshop to teach ExM hands-on in August 2016. Given the potential for ExM to solve many problems in neuroscience, we now propose to increase its power and versatility. Specifically, we will (Aim 1) develop optimized forms of ExM for difficult specimens (such as C. elegans), as well as strategies for single-sample validation (by creating ?physical scalebars? within samples), (Aim 2) invent new chemistries for expanding specimens by 20x or 80x in linear dimension, enabling ~15 nm and ~3 nm effective resolutions respectively, and (Aim 3) extend ExM anchoring chemistries for the visualization of lipids and DNA, as well as combinations of biomolecules (e.g., seeing proteins, DNA, and RNA all at once). Our project will result in tools of great applicability in neuroscience, as well as throughout biology. We propose a fast-paced, 4 year technology development grant that will result in tools that will enable a large number of scientific problems to be analyzed. We will distribute all tools as freely as possible, and teach usage thereof.